Characterization of cytochrome P450 enzymes involved in drug oxidations in mouse intestinal microsomes

1. Cytochrome P450 (P450, CYP) enzymes involved in drug oxidations in mouse intestines were characterized for their role in the first-pass metabolism of xenobiotics. 2. Preparation of mouse intestinal microsomes using a buffer containing glycerol and protease inhibitors including (p-amidinophenyl) methanesulphonyl fluoride, EDTA, soybean trypsin inhibitor, aprotinin, bestatin and leupeptine gave the highest testosterone 6beta-hydroxylase activity among several preparation buffers tested in this study. Testosterone 6beta-hydroxylase activity catalysed by mouse intestinal microsomes subjected to freezing and thawing was lower than that catalysed by unfrozen intestinal microsomes. 3. Low but significant catalytic activities of nifedipine oxidation, midazolam 1'- and 4-hydroxylation, chlorzoxazone 6-hydroxylation, bufuralol 1'- and 6-hydroxylations and tolbutamide methylhydroxylation were observed in mouse intestinal microsomes. Testosterone 6beta-hydroxylation, chlorzoxazone 6-hydroxylation, and bufuralol 1'- and 6-hydroxylations were inhibited by ketoconazole, diethyldithiocarbamate and quinine respectively. 4. Immunoblot analysis using anti-rat CYP3A antibodies demonstrated two immunoreactive bands showing similar migration in mouse intestinal and hepatic microsomes, although studies using anti-CYP1A, anti-CYP2C, anti-CYP2D and anti-CYP2E1 antibodies did not detect any band in mouse intestinal microsomes. 5. The results suggest that mouse intestinal microsomes should be prepared with glycerol and several protease inhibitors and that Cyp3a enzymes probably play an important role in drug oxidations catalysed by mouse intestine.     

Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

Homozygous deletion (homo-d) of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene is frequently found in malignant pleural mesothelioma (MPM). Fluorescence in situ hybridization (FISH) is commonly used to detect chromosomal deletion, and sometimes reveals more frequent heterozygous deletion (hetero-d) compared with homo-d. In clinical practice, such CDKN2A FISH results belong to the ‘borderline’ homo-d rate, which makes it difficult to definitively diagnose MPM. Microdeletion, [<200 kilobase (kb)], can induce a ‘pseudo’ hetero-d signal in FISH assays with long probes owing to redundant probe reactivity. Thus, the present study hypothesized that shorter FISH probes can effectively detect the small deletion status of the CDKN2A gene and increase homo-d rate in MPM, which has high hetero-d and low homo-d status. The present study aimed to evaluate the effectiveness of a shorter CDKN2A FISH probe in diagnosing MPM. CDKN2A FISH with either a 222 kb long probe (L-probe) or a 57 kb short probe (S-probe) was performed in four MPM cases with high hetero-d and low homo-d patterns. Furthermore, immunohistochemistry for methylthioadenosine phosphorylase (MTAP) and quantitative (q)PCR analyses were performed to confirm the microdeletion of the 9p21 locus. The results demonstrated that all four MPM cases retained MTAP protein expression. CDKN2A FISH with L-probe revealed high hetero-d (cases 1–4; 73.3, 37.1, 59.2 and 64.8%, respectively) and low homo-d (cases 1–4; 12.1, 12.4, 25.4 and 22.2%, respectively). CDKN2A FISH with S-probe revealed high homo-d (cases 1–4; 96.8, 90.0, 87.5 and 82.6%, respectively), with low hetero-d (cases 1–4; 0.0, 1.2, 1.2 and 4.3%, respectively). qPCR analysis demonstrated no allele deletions of the MTAP gene and two-allele deletions of the CDKN2A gene in 3/4 cases. Taken together, these results suggest that the S-probe detects the short homo-d of the 9p21 locus more effectively than the L-probe in MPM. This can assist in solving diagnostic difficulties in cases involving high hetero-d with low homo-d.     

Expression of phosphoprotein enriched in astrocytes 15 kDa (PEA-15) in astrocytic tumors: a novel approach of correlating malignancy grade and prognosis

Phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is a multifunctional protein that was first identified in brain astrocytes and that has subsequently been shown to be expressed in different tissues. Despite its many important roles, the clinical significance of PEA-15 expression levels in astrocytic tumors has yet to be properly defined. We studied the PEA-15 expression pattern of 65 patients [diagnosed according to World Health Organization (WHO) criteria] with diffuse astrocytoma (WHO grade II), anaplastic astrocytoma (grade III), and glioblastoma (grade IV). PEA-15 expression levels were immunohistochemically measured and categorized as no, low, or high expression. All tumors expressed PEA-15 in our study. Twenty-three (35.4%) and 42 (64.6%) tumors expressed low and high PEA-15 levels, respectively. In grade II astrocytoma (diffuse astrocytoma) and grade III astrocytoma (anaplastic astrocytoma), 100% and 88.9% of patients expressed high PEA-15 levels, respectively, while a smaller number (50%) of patients with grade IV astrocytoma (glioblastoma) expressed high PEA-15 levels. PEA-15 expression level was inversely associated with WHO grade (P = 0.0006). Next, we evaluated prognosis and PEA-15 expression levels in 43 patients with high-grade astrocytomas based on the following parameters: age, gender, WHO grade, surgical resection extent, MIB-1 labeling index (LI), and PEA-15 expression level. Multivariable analyses revealed that high PEA-15 expression level displayed a significant correlation with longer overall survival (OS) in high-grade astrocytomas (P = 0.0024). Patients with total resection survived significantly longer (P = 0.0044) than those with lower resection extent, while patients with MIB-1 labeling index ≤25% indicated significant (P = 0.0434) correlation with OS as well. In conclusion, PEA-15 expression level was inversely associated with WHO grade and may serve as an important prognostic factor for high-grade astrocytomas.     

Efficacy of epidermal growth factor receptor-targeted molecular therapy in anaplastic thyroid cancer cell lines

Anaplastic thyroid cancer is one of the most aggressive human malignancies and the outcomes of conventional therapy have been far from satisfactory. Recently, epidermal growth factor receptor (EGFR)-targeted therapy has been introduced as an alternative therapeutic strategy for highly malignant cancers. This study was undertaken to investigate the expression of EGFR in anaplastic thyroid cancer cell lines, and to explore the potential of therapies targeting EGFR as a new therapeutic approach. EGFR was universally expressed in anaplastic cancer cell lines at a variety of levels. Specific EGFR stimulation with epidermal growth factor showed significant phosphorylation of ERK1/2 and Akt, and resulted in marked growth stimulation in an anaplastic thyroid cancer cell line, which highly expressed EGFR. This EGFR-transmitted proliferation effect of the cancer cell line was completely inhibited by gefitinib, an EGFR tyrosine kinase inhibitor. Moreover, growth of xenografts inoculated in mice was inhibited in a dose-dependent manner with 25-50 mg kg(-1) of gefitinib administrated orally. Inhibition of EGFR-transmitted growth stimulation by gefitinib was clearly observed in anaplastic thyroid cancer cell lines. Our results suggested that EGFR-targeted therapy, such as gefitinib, might be worth further investigation for the treatment of anaplastic thyroid cancer.     

Real-world effectiveness of third- or later-line treatment in Japanese patients with HER2-positive,unresectable, recurrent or metastatic gastric cancer: a retrospective observational study

Background

Real-world evidence on the preference for and effectiveness of third- or later-line (3L +) monotherapy for HER2-positive gastric cancer is limited in Japan. This study evaluated the utility of nivolumab, irinotecan, and trifluridine/tipiracil (FTD/TPI) monotherapy as 3L + treatment in Japanese patients with HER2-positive gastric/gastroesophageal junction (G/GEJ) cancer who were previously treated with trastuzumab.

Methods

In this multicenter, retrospective, observational study (20 centers), data of eligible patients were extracted from medical records (September 22, 2017–March 31, 2020), with follow-up until June 30, 2020. Outcomes included overall survival (OS), real-world progression-free survival (rwPFS), time to treatment failure (TTF), objective response rate (ORR; complete response [CR] + partial response [PR]), and disease control rate (DCR).

Results

Of 127 enrolled patients, the overall analysis population comprised 117 patients (median [range] age, 71 [38–89] years). The most commonly prescribed 3L + monotherapy was nivolumab (n = 100), followed by irinotecan (n = 12) and FTD/TPI (n = 5). The median (95% confidence interval [CI]) OS, rwPFS, and TTF were 6.2 (4.5–8.0), 1.9 (1.5–2.3), and 1.8 (1.5–2.2) months, respectively, at median (range) 150 (25–1007) days of follow-up. The ORR (CR + PR) and DCR were 9.0% (1% + 8%) and 32.0%, respectively. Factors such as higher neutrophil–lymphocyte ratio (≥ 2.54), Glasgow prognostic score (≥ 1), Eastern Cooperative Oncology Group performance status (ECOG PS; ≥ 2), and hepatic metastasis significantly impacted OS.

Conclusions

The observed OS in this study for HER2-positive G/GEJ cancer was shorter than that reported previously, suggesting that the effectiveness of nivolumab, irinotecan, or FTD/TPI as 3L + therapy may be limited.

     

Prevention of halothane-induced hepatotoxicity by hemin pretreatment: protective role of heme oxygenase-1 induction

Reductive metabolism of halothane in phenobarbital-pretreated rats is known to increase free radical formation that results in hepatotoxicity. It also is associated with a marked induction of microsomal heme oxygenase-1 (HO-1), suggesting that there is an alteration in heme metabolism. In this study, we examined heme metabolism in rats pretreated with phenobarbital, followed by exposure to halothane-hypoxia. In this model, there was a significant decrease in microsomal cytochrome P450 content in the liver, followed by a rapid increase in free heme concentration and a decrease in the level of mRNA for the nonspecific delta-aminolevulinate synthase. A transient but dramatic induction of HO-1 mRNA and a prolonged induction of heat shock protein 70 mRNA also occurred. The HO-1 protein was detected principally in the hepatocytes around the central vein. Serum alanine transaminase (ALT) activity, an indicator of hepatic dysfunction, increased continuously throughout the experiment. Hemin pretreatment induced hepatic HO-1 with abrogation of the halothane-induced hepatotoxicity in this model, as judged by ALT activity and normal histology. Our findings in this study thus indicate that halothane-induced hepatotoxicity is due not only to its reductive metabolite formation, but also to an increase in hepatic free heme concentration, which is a potent prooxidant; HO-1 induction is an important protective response against such changes. This is also the first study to demonstrate that hemin pretreatment, which induces HO-1 prior to exposure to halothane, effectively prevents halothane-induced hepatotoxicity.     

Propranolol suppresses gastric cancer cell growth by regulating proliferation and apoptosis

Background

Despite improvements in gastric cancer treatment, the mortality associated with advanced gastric cancer is still high. The activation of β-adrenergic receptors by stress has been shown to accelerate the progression of several cancers. Accordingly, increasing evidence suggests that the blockade of β-adrenergic signaling can inhibit tumor growth. However, the effect of β-blockers, which target several signaling pathways, on gastric cancer remains to be elucidated. This study aimed to investigate the anti-tumor effects of propranolol, a non-selective β-blocker, on gastric cancer.

Methods

We explored the effect of propranolol on the MKN45 and NUGC3 gastric cancer cell lines. Its efficacy and the mechanism by which it exerts anti-tumor effects were examined using several assays (e.g., cell proliferation, cell cycle, apoptosis, and wound healing) and a xenograft mouse model.

Results

We found that propranolol inhibited tumor growth and induced G1-phase cell cycle arrest and apoptosis in both cell lines. Propranolol also decreased the expression of phosphorylated CREB-ATF and MEK-ERK pathways; suppressed the expression of matrix metalloproteinase-2, 9 and vascular endothelial growth factor; and inhibited gastric cancer cell migration. In the xenograft mouse model, propranolol treatment significantly inhibited tumor growth, and immunohistochemistry revealed that propranolol led to the suppression of proliferation and induction of apoptosis.

Conclusions

Propranolol inhibits the proliferation of gastric cancer cells by inducing G1-phase cell cycle arrest and apoptosis. These findings indicate that propranolol might have an opportunity as a new drug for gastric cancer.

     

The prevalence of human papillomavirus in oral premalignant lesions and squamous cell carcinoma in comparison to cervical lesions used as a positive control

Background  

Previous reports concerning the prevalence of human papillomavirus (HPV) in oral squamous cell carcinoma (OSCC) have observed varied results. The aim of this study was to evaluate the prevalence of HPV in oral premalignant lesions (OPL) and OSCC. For accurate HPV detection in oral lesions, comparative analysis was performed on cervical lesions as positive controls.     

Quantitative analysis of sodium fast and slow component in in vivo human brain tissue using MR Na image]

In vivo sodium concentrations in the normal brain tissue and a tumorous tissue were analyzed using MR Na image. The nuclear magnetic resonance enabled us to divide the signal from sodium in the living tissue into 2 parts based on the differences of T2 value. Those are fast component having the T2 value of less than 5 msec and slow component of 15-40 msec. We investigated the effect of macromolecules on T2 value of sodium image using polyvinyl alcohol (PVA) powder. MR Na image was taken with the parameters of TR/TD, 110 ms/1.9 ms (FID image) and TR/TE, 110 ms/20 ms (SE image). Saline solution showed high intensity on both FID image and SE image. Saline solution added PVA (PVA phantom) also showed high intensity on FID image, whereas the signal intensity of PVA phantom in SE image extinguished. To know the relation between the signal intensity and sodium concentration, sodium concentration--signal intensity curve was obtained using phantoms with various sodium concentrations (0.05-1.0%). This curve showed a direct proportion between sodium concentration and signal intensity on Na image. We measured further the sodium concentrations of the human brain tissue. Sodium phantoms were arranged around the heads and the MR Na images of the normal brains from 3 volunteers and a patient with a brain tumor (meningioma) were taken. The sodium concentrations of occipital lobe, basal ganglia and the tumorous tissue were calculated using the sodium concentration--signal intensity curve obtained from the phantoms arranged around the heads.(ABSTRACT TRUNCATED AT 250 WORDS)     

Wound,pressure ulcer and burn guidelines – 4: Guidelines for the management of connective tissue disease/vasculitis-associated skin ulcers

The Japanese Dermatological Association prepared guidelines focused on the treatment of skin ulcers associated with connective tissue disease/vasculitis practical in clinical settings of dermatological care. Skin ulcers associated with connective tissue diseases or vasculitis occur on the background of a wide variety of diseases including, typically, systemic sclerosis but also systemic lupus erythematosus (SLE), dermatomyositis, rheumatoid arthritis (RA), various vasculitides and antiphospholipid antibody syndrome (APS). Therefore, in preparing the present guidelines, we considered diagnostic/therapeutic approaches appropriate for each of these disorders to be necessary and developed algorithms and clinical questions for systemic sclerosis, SLE, dermatomyositis, RA, vasculitis and APS.